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Global Investigation of an Engineered Nitrogen-Fixing Escherichia Coli www.nature.com/scientificreports OPEN Global investigation of an engineered nitrogen-fxing Escherichia coli strain reveals Received: 18 December 2017 Accepted: 6 July 2018 regulatory coupling between host Published: xx xx xxxx and heterologous nitrogen-fxation genes Zhimin Yang1,2, Yunlei Han2, Yao Ma2, Qinghua Chen2, Yuhua Zhan2, Wei Lu2, Li Cai1, Mingsheng Hou1, Sanfeng Chen3, Yongliang Yan2 & Min Lin2 Transfer of nitrogen fxation (nif) genes from diazotrophs to amenable heterologous hosts is of increasing interest to genetically engineer nitrogen fxation. However, how the non-diazotrophic host maximizes opportunities to fne-tune the acquired capacity for nitrogen fxation has not been fully explored. In this study, a global investigation of an engineered nitrogen-fxing Escherichia coli strain EN-01 harboring a heterologous nif island from Pseudomonas stutzeri was performed via transcriptomics and proteomics analyses. A total of 1156 genes and 206 discriminative proteins were found to be signifcantly altered when cells were incubated under nitrogen-fxation conditions. Pathways for regulation, metabolic fux and oxygen protection to nitrogenase were particularly discussed. An NtrC-dependent regulatory coupling between E. coli nitrogen regulation system and nif genes was established. Additionally, pentose phosphate pathway was proposed to serve as the primary route for glucose catabolism and energy supply to nitrogenase. Meanwhile, HPLC analysis indicated that organic acids produced by EN-01 might have negative efects on nitrogenase activity. This study provides a global view of the complex network underlying the acquired nif genes in the recombinant E. coli and also provides clues for the optimization and redesign of robust nitrogen-fxing organisms to improve nitrogenase efciency by overcoming regulatory or metabolic obstacles. In nature, a variety of genes and islands can be rapidly and frequently horizontally transferred among bacteria, resulting in the acquisition of certain properties such as nitrogen fxation, antimicrobial resistance and patho- genesis, which help bacteria to succeed in altered habitats or new niches1–3. However, newly acquired genes or islands become a burden for bacteria if they are not properly integrated with host regulatory systems. A greater understanding of the physiological alterations that occur in an engineered cell following the insertion of large fragments of foreign DNA, especially from distant species, is needed to pave the way towards the goal of biolog- ical engineering. Biological nitrogen fxation is catalyzed, in most cases, by the molybdenum nitrogenase encoded by a highly conserved nifHDK gene cluster. Previous studies have shown that the nif genes encoding active nitrogenase can be transferred to non-nitrogen-fxing prokaryotes to impart the ability to reduce atmospheric nitrogen gas into ammonia as a nitrogen source4–12. From the perspective of synthetic biology, one key goal of studying biological 1Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China. 2National Key Facility for Crop Gene Resources and Genetic Improvement, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. 3State Key Laboratory of Agrobiotechnology and College of Biological Science, China Agricultural University, Beijing, 100193, China. Correspondence and requests for materials should be addressed to Y.Y. (email: yanyongliang@caas. cn) or M.L. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:10928 | DOI:10.1038/s41598-018-29204-0 1 www.nature.com/scientificreports/ nitrogen fxation is to facilitate the introduction of this ability into organisms of great importance for human beings, for instance, engineering autonomous nitrogen-fxing cereal crops13–15. Eventually, a successfully engi- neered N2-fxing non-legume crop may signifcantly cut down the use of chemical fertilizers for a cleaner envi- ronment and higher yields16,17. In recent years, the synthesis of nitrogen-fxing systems has become increasingly common due to advances in synthetic biology. Normally, a functional entity or pathway is frst detected in prokaryotes before being trans- ferred to eukaryotes and plants. Because of its well-studied genetic background, Escherichia coli is frequently used as the preferred frst-step research model. Following the pioneering work on nitrogen-fxation engineering in 1970s4,5, several groups have reported successful gene transfer of nif genes to E. coli in the past fve years11,12,18,19. However, these recombinant E. coli stains showed much lower nitrogenase activity compared with the original host11,12,, and the horizontally acquired ability was insufcient to enable diazotrophic growth on nitrogen-free medium, implying the presence of (i) regulatory coupling between the host and heterologous nitrogen-fxation systems, as well as (ii) a regulatory/or metabolic barrier that results in reduced nitrogenase activity in the engi- neered cells. To date, how the non-diazotrophic host maximizes opportunities to fne-tune the acquired capacity for nitrogen fxation has not yet been fully explored. Pseudomonas stutzeri A1501 is a root-associated bacterium that exhibits an unusual feature, for a Pseudomonas strain, the ability to fx nitrogen20–24. Te P. stutzeri A1501 genome contains a 49-kb nitrogen fxation island (NFI) that comprises the largest group of nif genes identifed to date25. Within this island, a total of 52 nif-related genes are organized into 11 putative NifA-δ54-dependent operons24. nif gene expression in A1501 was revealed to be tightly regulated at both the transcriptional and post-transcriptional levels22,23,26,27. Given its natural integrity and well-studied regulation, the A1501 NFI is a promising model for studying the synthetic biology of nitrogen fxation systems. We previously transferred the entire P. stutzeri A1501 NFI into E. coli and found that the nitrogenase activity of the engineered E. coli strain was dependent on the external ammonium concentration, oxygen tension and temperature12. Similar to previous reports, the nitrogenase activity of recombinant E. coli strain EN-01 was much lower than that of A1501. In the present study, to better understand the global regulatory efect of the host on NFI expression, we monitored the global transcriptional and proteomic profles of recombinant E. coli grown anaer- obically under nitrogen-fxation and nitrogen-repression conditions. Furthermore, the metabolic fux shif of E. coli EN-01 under nitrogen-fxation conditions was also determined by HPLC. To the best of our knowledge, this is the frst report of a global investigation into the regulatory cascade of nif genes in an engineered nitrogen-fxing organism. Tese data are particularly useful for providing a more comprehensive understanding of how the E. coli host intervenes in the transcriptional regulation of the “foreign” NFI to support a functional nitrogenase complex. Our results will also help guide eforts to more successfully remodel and optimize similar systems in other species. Results Overview of the E. coli EN-01 transcriptome and proteome under nitrogen-fxation and nitro- gen-repression conditions. Te expression of 1156 genes was signifcantly altered (≥2-fold, P ≤ 0.05) under nitrogen-fxation conditions compared with nitrogen-repression conditions, including 789 up-regulated and 367 down-regulated genes. Te up-regulated genes mainly belonged to three major functional categories: nitrogen metabolism, transport or membrane protein and unknown function, while the down-regulated genes were mainly involved in energy synthesis, transport, protein synthesis and regulation. Tese altered genes were further classifed according to the COG functional classifcation system. As shown in Fig. 1, 259 genes, of which 102 were down-regulated and 157 were up-regulated, were involved in bacterial preservation and processing of genetic information (DNA replication, duplication, repair and gene transcription, expression, etc.). A total of 361 genes (31% of the total altered genes) were involved in transport and metabolic pathways. An additional 90 genes were involved in energy synthesis and transformation processes, with 67 genes induced under nitrogen-fxation conditions (Fig. 1A). Moreover, the expression of 151 genes encoding proteins of unknown function was also signifcant altered. Transcriptome analysis has revealed that 255 genes were significantly up-regulated and 294 genes were severely down-regulated in P. stutzeri A1501 under nitrogen-fxation conditions25. Subsequent comparison of the E. coli EN-01 and P. stutzeri A1501 transcriptomes revealed that at least 24 genes in the E. coli EN-01 tran- scriptome showed a similar expression pattern to that in P. stutzeri A1501 (Supplementary Table S1), including the glnK-amtB operon, the two-component regulatory system ntrBC andthe serine protein kinase-coding gene prkA, implying that the overlapping genes or systems might be essential for NFI expression in E. coli. Proteomic analysis of E. coli EN-01 was also performed under identical conditions. A total of 110 protein spots increased by more than 2-fold under nitrogen-fxation conditions; notably, 55 proteins were only expressed under nitrogen-fxation conditions. In addition, 96 proteins were signifcantly down-regulated under nitrogen-fxation conditions, and the expression of 24 protein spots was abolished on the 2D-PAGE gel (Supplementary Fig. S1). A total of 138 proteins were
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